LncRNA PLCD3-OT1 Functions as a CeRNA to Prevent Age-Related Cataract by Sponging miR-224-5p and Regulating PLCD3 Expression

Abstract

Purpose: Long noncoding RNAs (lncRNAs) are important in disease progression and cellular functions. This study aimed to conduct global lncRNA profiling and characterize the role of lncRNA 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase delta 3-sence RNA 1 (PLCD3-OT1) in the progression of age-related cataract (ARC).

Methods: We performed lncRNA expression profiling of lens capsule from ARC groups and age-matched groups using high-throughput RNA-sequencing. Real-time PCR was conducted to detect the expression pattern of lncRNA and mRNA in the clinical samples and cell model. Assays of cell-counting kit-8, 5′-ethynyl-2′-deoxyuridine, TUNEL, and propidium iodide staining were used to detect cell viability, proliferation, apoptosis, and cell cycle. We also performed fluorescence in situ hybridization assay to detect the location of lncRNA, and verified the endogenous competitive RNA mechanism between miRNAs, lncRNAs, and target genes via double-luciferase reporter analyses.

Results: The expression of lncRNA PLCD3-OT1 and PLCD3 were significantly decreased in ARC. PLCD3-OT1 overexpression promoted the expression of PLCD3, cell viability, proliferation, and inhibited cell apoptosis upon oxidative stress, while knockdown of PLCD3 showed the opposite results. Mechanistically, PLCD3-OT1functions through positively regulation the expression of PLCD3. In addition, PLCD3-OT1 may act as a ceRNA to regulate the expression of PLCD3 through competition for miR-224-5p.

Conclusions: PLCD3-OT1 and PLCD3 may become potential therapeutic targets for the prognosis, diagnosis, and treatment of ARC.

Age-related cataract (ARC), characterized by lens opacification, is the most common cause of severe visual impairment and blinding, as well as one of the greatest worldwide public health challenges of the 21st century.¹ ARC is an age-related disease caused by multiple pathogenic factors. Age, sex, smoking, sunlight exposure, genetic predisposition, heavy drinking, and cardiovascular factors may contribute to ARC progression.^2,3 There are three types of ARC, cortical (C), nuclear (N), and posterior subcapsular (PSC), based on the degenerated region of the lens. Cataract surgery is the only available treatment that can significantly improve the refractive outcome of patients, but surgery has a high cost and can lead to postsurgical complications, such as posterior capsular opacification.^4–6 

The precise mechanisms of ARC are not completely understood, but oxidative stress is known to play an important role in disease pathogenesis. Lens epithelial cells (LECs), a thin layer of cells in the anterior lens capsule, are primarily responsible for the organ's defense against oxidative stress. Many morphologic and functional changes occur in LECs during cataract formation, including increased proteolysis, an altered cell cycle, DNA damage, and changes in growth and differentiation of LECs.⁷ 

Long noncoding RNAs (lncRNAs) are widely present in living organisms. These molecules, whose length is more than 200 bp, can regulate gene expression at the epigenetic, transcriptional, and posttranscriptional levels. Therefore, lncRNAs can influence cell proliferation, apoptosis, vitality, immune response, and oxidative stress. There are seven types of lncRNAs, including sense lncRNAs, antisense lncRNAs, intronic lncRNAs, bidirectional lncRNAs, intergenic lncRNAs (lincRNAs), enhancer RNAs (eRNAs), and circular RNAs (circRNAs) based on their genomic organization or their relationship to protein coding genes.⁸ 

Aberrant lncRNA expression is associated with several human disorders, such as cancer, obesity, and cardiovascular diseases.^9–11 Recently, accumulating evidence has revealed that lncRNAs are related to the occurrence and development of ocular diseases.^12–14 However, the role of lncRNAs in the oxidative stress of ARC remains unclear. 

Therefore, we hypothesized that aberrant lncRNA expression in ARC patients interrupts oxidative damage and repair pathways and contributes to the development of ARC. To test this hypothesis, we determined the lncRNA and mRNA expression profiles in human capsulorhexis samples by high-throughput sequencing, and lncRNAs were found to be novel expression signatures modulating cell vitality, proliferation, and apoptosis in the ARC group. 

Materials and Methods

Study Participants

This study was approved by the Ethics Committee of the Affiliated Hospital of Nantong University (China) and carried out in accordance with the principles of the Declaration of Helsinki. We explained the purpose and procedures of the study to all participants, and all participants signed informed consent. 

Twelve patients with ARNC, 12 patients with ARCC, and 12 patients with PSC cataract were included according to the Lens Opacities Classification III (LOCS III)¹⁵ classification. In addition, 16 age-matched individuals with vitreoretinal diseases, who had transparent lenses extracted, were taken as controls. Patients with complicated cataracts because of high myopia, ocular injury, diabetes, ocular inflammation, or glaucoma and patients with systematic diseases, such as hypertension and diabetes, were excluded from the study. The demographic information of study participants was for listed in Supplementary Table S1. 

All lens capsules were obtained intact by means of continuous curvilinear capsulorhexis. Care should be taken to avoid vascular contact or damage to the iris or other intraocular structures. The samples were then divided into two parts. Some of them was used to extract RNA and mixed with QIAzol Lysis Reagent rapidly, then stored them at −80°C for later use. The other part was used to extract protein and was frozen directly and preservation at −80°C for future use. 

RNA Extraction, Data Analysis of RNA-Sequencing

According to the manufacturer's instructions (Qiagen, Dusseldorf, Germany), total RNA was extracted using a miRNeasy mini kit. The Agilent 2200 Bioanalyzer (Agilent, Palo Alto, USA) was used to measure RNA quality. The analyzer produced an RNA integrity number (RIN) between one and 10, of which 10 was the highest quality level, showing the least degradation. Sequencing was performed at Guangzhou RiboBio Co., Ltd., with an Illumina HiSeq 4000 system (Santiago, CA, USA). The RNA-seq data were analyzed by Shanghai Biochip (Shanghai, China). 

Cell Culture and UVB Irradiation

Human lens epithelium cell line (SRA011/04) was cultured in culture medium constituted by Dulbecco's modified Eagle's medium (Invitrogen-Gibco, Carlsbad, CA, USA), 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), and 100 U/mL penicillin. Cells were passaged at 70% to approximately 80% confluency. All experiments used cells within six generations of the original cells. 

The method of UVB exposure and detail information about UVB lamp were on the basis of our previous paper.³ What is more, the exposure time was 15 minutes. 

cDNA Preparation and Relative Quantification of mRNA Levels

Complementary (c)DNA was synthesized using an RT reagent kit (Thermo, Waltham, MA, USA), SYBR Green Master Mix (Roche, Basel, Switzerland), and a Step One Plus Real-Time PCR System (ABI, Carlsbad, CA, USA) was used to perform qRT-PCR. The cycle threshold (CT) values were normalized to that of a housekeeping gene in the same sample, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. The fold change of relative gene expression was determined using the comparative CT (2^−ΔΔCT) method. 

The cDNAs of miRNAs were synthesized using the Bulge-Loop miRNA RT kit (RiboBio Co., Guangzhou, China), and qRT-PCR was performed with the Bulge-Loop miRNA qRT-PCR kit (RiboBio Co.) to measure the expression of mature miR-224-5p /miRNA 296-5p in cells using the Step One Plus Real-Time PCR System (ABI). The cycle number for the fluorescent signal in each reaction tube was used to determine the relative amount of miR-224-5p/miRNA 296-5p using the 2^−ΔΔCT method. U6 was used as an endogenous control. The primers for qRT-PCR were listed in Supplementary Table S2. 

Western Blot Assays

At 24 hours after UVB irradiation, total protein was extracted from cells. Detail steps of Western blot assays were described in our previous article,^3,16 and the primary antibodies are shown in the Table. Alkaline phosphatase-conjugated goat anti-rabbit and an anti-mouse IgG antibody (1:10,000; Santa Cruz, Dallas, TX, USA) were used as secondary antibodies. 

Table

 

The Information of Antibodies Used in the Study

 

Fluorescence In Situ Hybridization (FISH) Assay

Cells grown on coverslips were fixed with 4% paraformaldehyde at 25°C for 15 minutes, washed three times with PBS, and treated with 0.5% Triton X-100 at room temperature for 10 minutes. The samples were dehydrated in 3% H₂O₂ and air-dried. After probe hybridization solution was added, the samples were mounted, denatured at 88°C for 5 minutes, and hybridized in a humid and dark environment at 37°C for 16 to 72 hours with FITC-labeled PLCD3-OT1 probe (Exon Biotechnology Inc., Guangzhou, China). The samples were washed three times with a preheated (43°C) solution consisting of 50% formamide and 2× saline sodium citrate (SSC) and then washed twice with 2× SSC (37°C). After the samples were counterstained with 4′,6-diamidino-2-phenylindole, they were mounted with fluorescence mounting medium and imaged with a microscope. 

PLCD3-OT1 Overexpression and Cotransfection

For analyzing overexpression, the sequence of PLCD3-OT1 was cloned into the lentiviral expression vector LV5 (GenePharma, Shanghai, China), and lentiviruses (1 × 10⁹ TU mL⁻¹) were generated with Lentivector Expression Systems (Shanghai, China). SRA01/04 were inoculated into 6-well plates at the density of 5 × 10³ per well. After 24 hours of culture, the cells were infected with lentivirus that was constituted by 200-μL recombinant PLCD3-OT1 lentiviral stocks or control lentiviral stocks and 800-μL medium, and the time of infection was 24 hours. Two days later, green fluorescent protein observed by fluorescence microscopy can reflect the infection rate of the cells, directly. Then, infected cells were seeded into one 100-mm cell culture dish and cultured in the medium with 2 μg mL⁻¹ puromycin for 7 days until stable clones became obvious. Quantitative (q)PCR analysis was used to select PLCD3-OT1 overexpression cell lines (designated as OV) and control cell lines (designated as NC). Eventually, there were three paired NC and OV cell clones for our experiments. Because of the similar results, we showed only one pair in this article. 

PLCD3 small interfering (si)RNA and scrambled siRNA were supplied by RiboBio Co. (Guangzhou, China). Cell lines were seeded in volumes of 100 μL for 96-well plates and 500 μL for 24-well plates at a density of 1 × 10⁵ per well. The siRNA (50 nM) mixed with transfection reagent gently added to the cells. 

Cell Counting Kit-8 (CCK8) and TUNEL Assays

CCK8 assay (Dojindo, Kyushu Island, Japan) was used to detect the cell viability and TUNEL assay (Roche, Mannheim, Germany) was used to detect the cell apoptosis. There assays were carried out according to the manufacturer's protocol, and our previous article.³ 

EdU (5′-Ethynyl-2′-Deoxyuridine) Assay

After a 72-hur siRNA transfection and 24 hours after a 15-minute UVB exposure, cell proliferation was detected using an EdU labeling/detection kit (RiboBio Co., Guangzhou, China) in this study. Then, 50-μM EdU-labeling medium was added to the cell culture, which was incubated for 24 hours at 37°C under 5% CO₂. After the cells were washed with PBS, staining with anti-EdU working solution was performed at room temperature for 30 minutes. After the cells were washed with 0.5% Triton X-100 in PBS, they were incubated with 5 μg/mL Hoechst 33342 dye at room temperature for 30 minutes, followed by observation under a fluorescence microscope (Leica, Weztlar, Germany). 

Flow Cytometry (FCM)

Cell cycle analysis was performed by propidium iodide (PI) staining (BD, Franklin, NJ, USA). The cultured SRA01/04 cells that were infected or transfected were trypsinized and harvested in PBS, followed by washing with PBS. Then, the cells were resuspended in PBS at 1 × 10⁶/mL and aggregated into pellets by centrifugation. After the supernatant was discarded, 1 mL anhydrous ethanol was added, blended by vortexing, and incubated for 24 hours at 4°C. Then, the cells were washed three times with PBS, and after the supernatant was discarded, 300 μL of DNA staining solution was added, blended by vortexing, and incubated for 15 minutes at room temperature, followed by a FACSCalibur flow cytometric analysis (BD). 

Luciferase Reporter Assay

A luciferase reporter assay was performed using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA) to confirm the direct binding of PLCD3-OT1 and miR-224-5p/ miR-296-5p. First, the wild-type and mutant sequences of PLCD3-OT1 were subcloned into PsiCHECK2 luciferase reporter vectors. Then, cells were cotransfected with PsiCHECK2 vectors and miR-224-5p/miR-296-5p mimics or miRNA negative controls. The fluorescence signals were detected 48 hours after transfection according to the manufacturer's instructions, and the relative fluorescence value (luciferase/Renilla) was calculated. 

Statistical Analysis

Each experiment was repeated three times, and the data were expressed as Display Formula\(\def\upalpha{\unicode[Times]{x3B1}}\)\(\def\upbeta{\unicode[Times]{x3B2}}\)\(\def\upgamma{\unicode[Times]{x3B3}}\)\(\def\updelta{\unicode[Times]{x3B4}}\)\(\def\upvarepsilon{\unicode[Times]{x3B5}}\)\(\def\upzeta{\unicode[Times]{x3B6}}\)\(\def\upeta{\unicode[Times]{x3B7}}\)\(\def\uptheta{\unicode[Times]{x3B8}}\)\(\def\upiota{\unicode[Times]{x3B9}}\)\(\def\upkappa{\unicode[Times]{x3BA}}\)\(\def\uplambda{\unicode[Times]{x3BB}}\)\(\def\upmu{\unicode[Times]{x3BC}}\)\(\def\upnu{\unicode[Times]{x3BD}}\)\(\def\upxi{\unicode[Times]{x3BE}}\)\(\def\upomicron{\unicode[Times]{x3BF}}\)\(\def\uppi{\unicode[Times]{x3C0}}\)\(\def\uprho{\unicode[Times]{x3C1}}\)\(\def\upsigma{\unicode[Times]{x3C3}}\)\(\def\uptau{\unicode[Times]{x3C4}}\)\(\def\upupsilon{\unicode[Times]{x3C5}}\)\(\def\upphi{\unicode[Times]{x3C6}}\)\(\def\upchi{\unicode[Times]{x3C7}}\)\(\def\uppsy{\unicode[Times]{x3C8}}\)\(\def\upomega{\unicode[Times]{x3C9}}\)\(\def\bialpha{\boldsymbol{\alpha}}\)\(\def\bibeta{\boldsymbol{\beta}}\)\(\def\bigamma{\boldsymbol{\gamma}}\)\(\def\bidelta{\boldsymbol{\delta}}\)\(\def\bivarepsilon{\boldsymbol{\varepsilon}}\)\(\def\bizeta{\boldsymbol{\zeta}}\)\(\def\bieta{\boldsymbol{\eta}}\)\(\def\bitheta{\boldsymbol{\theta}}\)\(\def\biiota{\boldsymbol{\iota}}\)\(\def\bikappa{\boldsymbol{\kappa}}\)\(\def\bilambda{\boldsymbol{\lambda}}\)\(\def\bimu{\boldsymbol{\mu}}\)\(\def\binu{\boldsymbol{\nu}}\)\(\def\bixi{\boldsymbol{\xi}}\)\(\def\biomicron{\boldsymbol{\micron}}\)\(\def\bipi{\boldsymbol{\pi}}\)\(\def\birho{\boldsymbol{\rho}}\)\(\def\bisigma{\boldsymbol{\sigma}}\)\(\def\bitau{\boldsymbol{\tau}}\)\(\def\biupsilon{\boldsymbol{\upsilon}}\)\(\def\biphi{\boldsymbol{\phi}}\)\(\def\bichi{\boldsymbol{\chi}}\)\(\def\bipsy{\boldsymbol{\psy}}\)\(\def\biomega{\boldsymbol{\omega}}\)\(\def\bupalpha{\unicode[Times]{x1D6C2}}\)\(\def\bupbeta{\unicode[Times]{x1D6C3}}\)\(\def\bupgamma{\unicode[Times]{x1D6C4}}\)\(\def\bupdelta{\unicode[Times]{x1D6C5}}\)\(\def\bupepsilon{\unicode[Times]{x1D6C6}}\)\(\def\bupvarepsilon{\unicode[Times]{x1D6DC}}\)\(\def\bupzeta{\unicode[Times]{x1D6C7}}\)\(\def\bupeta{\unicode[Times]{x1D6C8}}\)\(\def\buptheta{\unicode[Times]{x1D6C9}}\)\(\def\bupiota{\unicode[Times]{x1D6CA}}\)\(\def\bupkappa{\unicode[Times]{x1D6CB}}\)\(\def\buplambda{\unicode[Times]{x1D6CC}}\)\(\def\bupmu{\unicode[Times]{x1D6CD}}\)\(\def\bupnu{\unicode[Times]{x1D6CE}}\)\(\def\bupxi{\unicode[Times]{x1D6CF}}\)\(\def\bupomicron{\unicode[Times]{x1D6D0}}\)\(\def\buppi{\unicode[Times]{x1D6D1}}\)\(\def\buprho{\unicode[Times]{x1D6D2}}\)\(\def\bupsigma{\unicode[Times]{x1D6D4}}\)\(\def\buptau{\unicode[Times]{x1D6D5}}\)\(\def\bupupsilon{\unicode[Times]{x1D6D6}}\)\(\def\bupphi{\unicode[Times]{x1D6D7}}\)\(\def\bupchi{\unicode[Times]{x1D6D8}}\)\(\def\buppsy{\unicode[Times]{x1D6D9}}\)\(\def\bupomega{\unicode[Times]{x1D6DA}}\)\(\def\bupvartheta{\unicode[Times]{x1D6DD}}\)\(\def\bGamma{\bf{\Gamma}}\)\(\def\bDelta{\bf{\Delta}}\)\(\def\bTheta{\bf{\Theta}}\)\(\def\bLambda{\bf{\Lambda}}\)\(\def\bXi{\bf{\Xi}}\)\(\def\bPi{\bf{\Pi}}\)\(\def\bSigma{\bf{\Sigma}}\)\(\def\bUpsilon{\bf{\Upsilon}}\)\(\def\bPhi{\bf{\Phi}}\)\(\def\bPsi{\bf{\Psi}}\)\(\def\bOmega{\bf{\Omega}}\)\(\def\iGamma{\unicode[Times]{x1D6E4}}\)\(\def\iDelta{\unicode[Times]{x1D6E5}}\)\(\def\iTheta{\unicode[Times]{x1D6E9}}\)\(\def\iLambda{\unicode[Times]{x1D6EC}}\)\(\def\iXi{\unicode[Times]{x1D6EF}}\)\(\def\iPi{\unicode[Times]{x1D6F1}}\)\(\def\iSigma{\unicode[Times]{x1D6F4}}\)\(\def\iUpsilon{\unicode[Times]{x1D6F6}}\)\(\def\iPhi{\unicode[Times]{x1D6F7}}\)\(\def\iPsi{\unicode[Times]{x1D6F9}}\)\(\def\iOmega{\unicode[Times]{x1D6FA}}\)\(\def\biGamma{\unicode[Times]{x1D71E}}\)\(\def\biDelta{\unicode[Times]{x1D71F}}\)\(\def\biTheta{\unicode[Times]{x1D723}}\)\(\def\biLambda{\unicode[Times]{x1D726}}\)\(\def\biXi{\unicode[Times]{x1D729}}\)\(\def\biPi{\unicode[Times]{x1D72B}}\)\(\def\biSigma{\unicode[Times]{x1D72E}}\)\(\def\biUpsilon{\unicode[Times]{x1D730}}\)\(\def\biPhi{\unicode[Times]{x1D731}}\)\(\def\biPsi{\unicode[Times]{x1D733}}\)\(\def\biOmega{\unicode[Times]{x1D734}}\)\(\bar \chi \) ± SD. SPSS 25.0 (IBM Corp, Armonk, NY, USA) was used to process and analyze all data. The single factor ANOVA was used for statistical analyses, and P values less than 0.05/0.01 were statistically significant. 

Results

Overview of the lncRNA and mRNA Expression Profiles

To identify ARC-related lncRNAs, we extracted total RNAs of the lens capsule from two patients with ARNC, two patients with ARCC, two patients with PSC ARC, and six age-matched transparent lenses. Then, the lncRNA expression profile was analyzed using a high-throughput RNA-sequencing analysis. As shown in Figure 1a, principal component analysis (PCA) of the ARC and control groups indicated that one of the control lens capsules had a large difference compared with the other control lens capsules, and thus, we selected six lens capsules from patients with ARC and five transparent lens capsules for the next study. A differential expression fold change more than 2 and q value less than 0.05 in the negative binomial distribution of the ARC and control groups were used as the selection criteria. A total of 182 lncRNAs were downregulated and 49 lncRNAs were upregulated in the lens capsules of patients with ARC (Fig. 1b). Additionally, 271 mRNAs were downregulated and 214 mRNAs were upregulated in the lens capsules of patients with ARC (Fig. 1d). To reveal the potential role of lncRNAs in ARC, we performed gene enrichment analysis to determine the gene and gene product enrichment in biological processes, cellular components, and molecular functions. The biological functions of the differentially expressed genes were related to inflammation, immunity, extracellular matrix, signal transduction, and cell differentiation. The cellular components were related to the extracellular matrix and membrane, and the molecular functions were related to oxygen transporter activity and calcium binding (Fig. 1f). 

Figure 1

 

Identification of lncRNAs in ARC using high-throughput sequencing analysis. (a) PCA of lncRNAs in the ARC and control groups; one of the control lens capsules had a large difference compared with the other control lens capsules. (b) Volcano plots of differentially expressed lncRNAs in the ARC and control groups. Criteria for selecting different lncRNAs: fold change >2 and q value < 0.05. Blue points: downregulated lncRNAs in ARC; red points: upregulated lncRNAs in ARC. (c) Heat map and hierarchic cluster analysis of differentially expressed lncRNAs in the ARC and control groups. Red: downregulated lncRNAs in ARC; blue: upregulated lncRNAs in ARC. (d) Volcano plots of differentially expressed mRNAs in the ARC and control groups. Criteria for selecting differentially expressed mRNAs: fold change >2 and q value < 0.05. Blue points: downregulated mRNAs in ARC; red points: upregulated mRNAs in ARC. (e) Heat map and hierarchical cluster analysis of differentially expressed mRNAs in the ARC and control groups. Red: downregulated mRNAs in ARC; blue: upregulated mRNAs in ARC. (f) The Gene Ontology (GO) enrichment analysis provided a controlled vocabulary to describe the differentially expressed lncRNAs-coexpressed mRNAs. The GO analysis covered the following three domains: biological processes, cellular components, and molecular functions (P < 0.05).

PLCD3-OT1 and PLCD3 Were Downregulated in ARC

Among the different lncRNAs, lncRNA NONHSAT176260.1, which overlapped 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase delta-3 (PLCD3) on the sense strand, was identified (Supplementary Fig. S1). The main reason was that phospholipase C (PLC) can play a protective role in myocardial oxidative injury,¹⁷ and this lncRNA showed the most dramatic change. In addition, according to the guidelines on gene nomenclature of the Human Genome Organization, we renamed NONHSAT176260.1 as PLCD3-OT1, which it is called hereafter. As shown in Figure 2a, we used qRT-PCR to validate the expression of PLCD3-OT1, and PLCD3-OT1 levels were considerably lower in the ARC group than in the control group (P < 0.001). PLCD3 mRNA (Fig. 2b) and protein (Figs. 2c, 2d) levels were also considerably lower in the ARC groups than in the control group (P < 0.001). 

Figure 2

 

PLCD3-OT1 and PLCD3 mRNA and protein expression in the capsular lenses of the ARC groups and the control group. (a) The expression of PLCD3-OT1 was lower in the ARC groups than in the control group (n = 10). QRT-PCR was used to determine the expression levels and the comparative CT (2^−ΔCT) method was used to calculated them. (b) PLCD3 mRNA levels for the ARC groups and the control group (n = 10). (c, d) PLCD3 protein levels for the ARC groups and the control group (n = 10). ***P < 0.001. C, cortical; N, nuclear; PSC, posterior subcapsular.

Cell Viability and Proliferation Were Increased, Apoptosis was Decreased, and Cell Cycle Changes Were Observed, After Overexpression of PLCD3-OT1 in SRA01/04 Cells

To investigate the role of PLCD3-OT1 in regulation of LECs function, SRA01/04 was infected with lentivirus encoding PLCD-OT1 or with control lentivirus. QRT-PCR analysis showed a 4-fold increase in PLCD3-OT1 expression in OV cells compared with the NC and control cells (Fig. 3a). We also investigated the expression of PLCD3. Figure 3b shows that the mRNA level in OV cells was three times higher than that in the NC and control groups. Western blot showed the same results (Figs. 3c, 3d). Overexpression of PLCD3-OT1 significantly increased the mRNA and protein levels of PLCD3. The cells were cultured for 24 hours after exposure to UVB for 15 minutes. Then, we detected the cell viability by CCK8 assays; as shown in Figure 4a, overexpression of PLCD3-OT1 significantly reduced the apoptosis caused by UVB radiation. The TUNEL assay showed the same results (Figs. 4b, 4c), and overexpression of PLCD3-OT1 significantly reduced the damage caused by UVB radiation (Figs. 4d, 4e). To determine whether PLCD3-OT1 induced SRA01/04 cell proliferation by regulating the cell cycle, we analyzed the cell cycle by PI staining after overexpression of PLCD3-OT1; as shown in Figures 4f and 4g, the OV group exhibited a shorter G0/G1 phase with a concomitantly longer S+G2/M phase than the NC group. Western blotting was used to detect Bax, Bad, Bcl-2, cyclin D1, and cyclin A2 (Figs. 4h, 4i). 

Figure 3

 

PLCD3 mRNA and protein and PLCD3-OT1 expression in SRA01/04 cells after overexpression of PLCD3-OT1. (a) QRT-PCR verification of the efficiency of PLCD3-OT1 overexpression. (b) PLCD3 mRNA expression was measured by qRT-PCR in SRA01/04 cells after overexpression of PLCD3-OT1. (c, d) PLCD3 protein expression was measured by Western blot analysis of SRA01/04 cells after overexpression of PLCD3-OT1, and the variation trend was similar to that of PLCD3-OT1. ***P < 0.001, **P < 0.01, *P < 0.05.

Figure 4

 

The cytologic change of SRA01/04 cells after overexpression of PLCD3-OT1. After exposure to UVB of 15 minutes, the cells were cultured for 24 hours. (a) The effect of overexpression of PLCD3-OT1 on cell viability in SRA01/04 was measured by CCK8 assays after UVB exposure. (b) Overexpression of PLCD3-OT1 decreased cell apoptosis as measured by TUNEL assays after UVB exposure; red, TUNEL staining; blue, Hoechst staining. (c) Population of TUNEL-positive cells. (d) Overexpression of PLCD3-OT1 increased cell proliferation and reduced the cell damage caused by UVB radiation; red, EdU staining; blue, Hoechst staining. (e) Population of EdU-positive cells. (f) OV showed a shorter G0/G1 phase with a concomitantly longer S+G2/M phase; (g) population of each phase in the cell cycle. (h, i) The relevant protein changed after overexpression of PLCD3-OT1. ***,#P < 0.001, *P < 0.05

PLCD3-OT1 and PLCD3 mRNA and Protein Levels Were Increased in SRA01/04 Cells Irradiated With UVB

The precise mechanisms of ARC are not completely understood, but oxidative stress is known to play an important role in the disease pathogenesis. To determine the role of PLCD3-OT1 and PLCD3 in the oxidative damage to the lens, we established an acute oxidative injury model of SRA01/04 cells. SRA01/04 cells were cultured for 24 hours after exposure to UVB for 15 minutes, the apoptosis of SRA01/04 cells was increased, and the proliferation was decreased compared with that of the control group. QRT-PCR showed that PLCD3-OT1 was increased (Fig. 5a), similar to the PLCD3 mRNA levels (Fig. 5b), and the protein expression of PLCD3 was also increased, as shown by Western blot analysis (Figs. 5c, 5d). 

Figure 5

 

PLCD3-OT1 and PLCD3 mRNA and protein levels in the SRA01/04 cells after UVB exposure for 15 minutes. (a) PLCD3-OT1 expression was measured by qRT-PCR in SRA01/04 cells after UVB exposure, and PLCD3-OT1 expression levels were significantly higher than that of the control group, which was calculated using the comparative CT (2^−ΔΔCT) method. (b) PLCD3 mRNA expression levels also increased significantly. (c, d) Increased PLCD3 protein expression was detected in the UVB group. ***P < 0.001, **P < 0.01.

Cell Viability and Proliferation Were Reduced, Apoptosis was Increased, and Cell Cycle Changed After Silencing PLCD3 in SRA01/04 Cells

To make clear whether PLCD3 has a marked impact on LECs function in vitro, SRA01/04 cells were transfected with siRNA to silence PLCD3. Then, total RNA and protein were extracted from SRA01/04 cells after 72 hours of incubation. As shown in Figures 6a and 6b, the knockdown efficiency of siRNA2 was approximately 80% for PLCD3 mRNA (Fig. 6a) and approximately 35% for PLCD3 protein (Figs. 6b, 6c); thus, this siRNA was selected to carry out the following experiments. We also detected the PLCD3-OT1 expression levels, and the variation trend was similar to that of PLCD3 (Fig. 6d). As shown in Figure 7a, cell viability was decreased in the siRNA2 group. When UVB was used to irradiate cells, the siRNA2+UVB group showed the lowest values (Fig. 7b). According to TUNEL assay, the cell apoptosis of the siRNA2 group was significantly different compared with that of the other groups (P < 0.001) (Figs. 7c, 7d). After UVB exposure, apoptosis was increased (Figs. 7e, 7f). The result of EdU assay showed PLCD3 knockdown led to a decrease in cell proliferation (Figs. 7g, 7h). To determine whether PLCD3 induced SRA01/04 cell proliferation by regulating the cell cycle, we analyzed the cell cycle distribution after transfection with siRNA2 by FCM. We also analyzed the cell cycle by PI staining. Compared with the control cells, SRA01/04 cells transfected with siRNA2 exhibited a longer G0/G1 phase with a concomitantly shorter S+G2/M phase (Figs. 7i, 7j). To further determine the effect of PLCD3 on the regulation of SRA01/04 cell apoptosis, proliferation, and cell cycle, we performed Western blotting to detect Bax, Bad, Bcl-2, cyclin D1, and cyclin A2 (Figs. 7k, 7l). These results suggested that knocking down PLCD3 inhibited SRA01/04 cell proliferation and increased cell apoptosis. 

Figure 6

 

PLCD3 mRNA and protein levels and PLCD3-OT1 expression in SRA01/04 cells after PLCD3 silencing. After exposure to UVB of 15 minutes, the cells were cultured for 24 hours. (a) QRT-PCR verification of the knockdown efficiency of three types of siRNAs targeting PLCD3; the knockdown efficiency of siRNA2 was the highest. QRT-PCR measured PLCD3 expression in SRA01/04 cells after siRNA transfection. (b, c) Western blot analyzed PLCD3 protein expression in SRA01/04 cells after siRNA transfection. (d) QRT-PCR measured PLCD3-OT1 expression in SRA01/04 cells after siRNA transfection, and the variation trend was similar to that of PLCD3. ***P < 0.001, *P < 0.05.

Figure 7

 

The cytologic change of SRA01/04 cells after PLCD3 silencing. (a) The effect of silencing PLCD3 on mitochondrial dehydrogenase activity in SRA01/04 cells was measured by CCK8 assays. (b) After UVB exposure, PLCD3 knockdown further decreased cell viability. (c) PLCD3 knockdown increased cell apoptosis as measured by TUNEL assays, (e) and the UVB+siRNA2 group showed the most cell apoptosis; green, TUNEL staining; blue, Hoechst staining. (d, f) Population of TUNEL-positive cells, ***P < 0.001, *P < 0.05. (g) PLCD3 knockdown decreased cell proliferation as measured by EdU assays, (h) population of EdU-positive cells, (i) SRA01/04 cells transfected with siRNA2 exhibited a longer G0/G1 phase with a concomitantly shorter S+G2/M phase, (j) population of each phase in the cell cycle. (k, l) The relevant protein changed after PLCD3 knockdown.

PLCD3-OT1 was Predominantly Localized in the Cytosol of SRA01/04

To determine the location of PLCD3-OT1 in SRA01/04, we performed RNA FISH analyses using FITC-labeled probes that recognize PLCD3-OT1. We observed fluorescent signals (green) in the cytoplasm (Fig. 8), suggesting that PLCD3-OT1 is located in the cytoplasm. 

Figure 8

 

PLCD3-OT1 cell location. According to the RNA FISH assay of PLCD3-OT1 in SRA01/04, PLCD3-OT1 was predominantly localized in the cytosol. FITC, green fluorescence; DAPI, blue fluorescence. PLCD3-OT1: FITC-labeled PLCD3-OT1 probes, Control: no probe.

PLCD3-OT1 and PLCD3 are Targets of miR-224-5p

Bioinformatics analysis of miRNA recognition sequences on PLCD3-OT1 revealed the presence of two miRNA binding sites. The wild-type or mutant PLCD3-OT1 cDNA was cloned downstream of the luciferase gene and named PLCD3-OT1-WT or PLCD3-OT1-Mut (Fig. 9a). These vectors were transfected into SRA01/04 cells with miR-224-5p mimics or miR-296-5p mimics, and miRNA-NC was used as the negative control. As shown in Figure 9b, the luciferase activity in the PLCD3-OT1-WT group overexpressing miR-224-5p was reduced by 55% compared with that of the PLCD3-OT1-Mut group overexpressing miR-224-5p. However, the luciferase activity in the PLCD3-OT1-WT group with overexpression of miR-296-5p was almost unchanged. Therefore, this assay demonstrated that only miR-224-5p can directly bind to PLCD3-OT1 and PLCD3 through miRNA recognition sites, and we hypothesized that PLCD3-OT1 is a competing endogenous RNA (ceRNA) that regulates PLCD3 expression by sponging miR-224-5p and influencing the progression of ARC. 

Figure 9

 

PLCD3-OT1 is a target of miR-224-5p and acts as a competing endogenous RNA to regulate PLCD3 expression. (a) Bioinformatics analysis revealed two putative miRNA binding sites in the PLCD3-OT1 and PLCD3 sequence. PLCD3-OT1-Mut was generated by mutating the putative miR-224-5p or miR-296-5p binding site. (b) Luciferase activity was determined using the dual luciferase assay and is shown as the relative luciferase activity normalized to Renilla activity. MiR-NC was used as the negative control.

miR-224-5p was Increased in ARC and Could Regulate the Expression of PLCD3-OT1 and PLCD3

To support our hypothesis, we detected the expression of miR-225-5p in the ARC group and control group. As shown in Figure 10a, miR-224-5p expression was considerably lower in the ARC group than in the control group (P < 0.001). Then, miR-224-5p mimics and inhibitors were transfected in SRA01/04 cells, and Figure 10b shows that we successfully constructed transfected cells. QRT-PCR revealed that overexpression of miR-225-5p decreased the expression levels of PLCD3-OT1 and PLCD3, and knockdown of miR-225-5p led to decreased expression levels of PLCD3-OT1 and PLCD3 (Figs. 10c, 10d). Western blot showed the same results (Figs. 10e, 10f). 

Figure 10

 

MiR-224-5p expression in the capsular lenses of the ARC groups and the control group and PLCD3-OT1 and PLCD3 mRNA and protein expression in SRA01/04 cells after transfection with miR-224-5p mimics or inhibitors. (a) The expression levels of miR-224-5p were higher in the ARC groups than in the control group (n = 10). QRT-PCR measured the expression levels and 2^−ΔCT method calculated them. (b) QRT-PCR verification of the transfection of miR-224-5p mimics and inhibitor. (c, d) PLCD3-OT1 and PLCD3 mRNA levels were measured by qRT-PCR, and the levels were all increased (decreased) after transfection with the miR-224-5p inhibitors (mimics). (e, f) Western blot was used to measure PLCD3 protein expression of SRA01/04 cells after transfection with miR-224-5p mimics or inhibitors. ***P < 0.001, *P < 0.05.

Discussion

LncRNAs, 15% of which are located in the cytoplasm,¹⁸ are usually expressed in the stage-specific manner of disease, tissue, or development, indicating specific functions for lncRNAs in development and diseases and indicating that these molecules are attractive therapeutic targets.^19–21 An increasing number of studies have shown that lncRNAs are involved in the pathogenesis of several ocular diseases, including corneal neovascularization,²² cataract,^23,24 glaucoma,²⁵ diabetic retinopathy,^22,26 proliferative vitreoretinopathy,^27,28 and ocular tumors.^29,30 

Although the mechanism of ARC is not completely understand, age, sex, smoking, sunlight exposure, genetic predisposition, heavy drinking, and cardiovascular factors may contribute to ARC progression.^2,3 UVB irradiation is one of the most important environmental risk factors for cataract formation.^31,32 What's more, some reports, including our studies, have shown UVB does not only induce the cortical cataract, but also has correlation with nuclear and posterior subcapsular cataract.^3,33–35 

UVB exposure may attack the oxidative pathways and generate ROS, causing oxidative modification of cellular macromolecules and accumulation of lipid peroxidation products,³ it also induces DNA lesion,³⁶ epigenetic modification,¹⁶ and generation of inflammatory media.³⁷ All these cellular effects of UVB irradiation finally promote cell apoptosis. UVB-induced cataract formation begins with damage to LECs, and apoptosis of LECs is an early event during cataract development.³² What is more, recently studies indicates apoptosis of LECs may be the cytologic foundation of all kinds of cataract except congenital cataract. 

In this study, we determined the expression profiles of lncRNAs and mRNAs in the ARC group and control group, and we also identified the function of PLCD3-OT1 in LECs by conducting loss- and gain-of-function studies. These results demonstrated that PLCD3-OT1 was downregulated in the ARC lens capsule compared with the control lens capsule and that PLCD3-OT1 overexpression protected LECs from UVB radiation, promoted LEC proliferation, and decreased LEC apoptosis. Moreover, to determine whether PLCD3-OT1 induced SRA01/04 cell proliferation with the regulation of the cell cycle, we assessed the cell cycle after PLCD3-OT1 overexpression. Cells with PLCD3-OT1 overexpression exhibited a shorter G0/G1 phase with a concomitantly longer S+G2/M phase than control cells. These results suggest that PLCD3-OT1 plays an important role in modulating the physiologic function of LECs and ARC progression, suggesting new research directions and indicating that PLCD3-OT1 is a therapeutic target in ARC. 

That lncRNAs have impact on the cellular function through various mechanisms, may be associated with the important role of lncRNAs in human disease, and the mechanisms are as follows: (1) lncRNA, as a competing endogenous (ce)RNA, binds to micro (mi)RNA; thus, it can increase the translation of corresponding genes, such as linc-MD1, which can regulate the expression of the muscle-specific gene transcription regulators MAML1 and MEF2C by binding to miRNA-206-133b.³⁸ (2) LncRNA binds directly to DNA methyltransferase (DNMT), and thus suppresses the methylation of promotor regions and promotes gene expression, such as lncRNA DBCCR1-003, which can inhibit DBCCR1 methylation by binding DNMT1; thus, it can upregulate DBCCR1 expression.³⁹ (3) LncRNAs can be a precursor of miRNAs and can be processed to form a miRNA to regulate gene expression; for example, H19 is a precursor of miRNA-675 and can enhance the proliferation and invasion of cancer cells in gastric cancer.¹⁰ (4) Antisense lncRNAs can downregulate gene expression by binding to target genes, such as SATB2-AS1, which can directly bind to SATB2 to increase cell proliferation and growth in osteosarcoma.⁴⁰ (5) LncRNAs can affect gene transcription and translation by regulating histone methylation; for example, HOTAIR, which can upregulate histone H3K29 demethylase, increases Snail expression and decreases PCDHB5 expression, enhancing the invasion of renal neoplasm cells.⁴¹ 

In this study, for the mechanism of PLCD3-OT1, subcellular location analysis of PLCD3-OT1 by RNA FISH assays demonstrated that PLCD3-OT1 was localized to the cytoplasm. Inspired by the ceRNA regulatory network, we speculated that the mechanism underlying PLCD3-OT1 regulation of LEC function is that lncRNA, as a ceRNA, binds to miRNAs to increase target gene expression. Therefore, we searched for potential interactions with miRNAs by bioinformatics analysis and luciferase assays to verify the direct binding ability of the predicted miRNA response elements on PLCD3-OT1 transcript. As was expected, we discovered that PLCD3-OT1 is the target of miR-224-5p. QRT-PCR analysis showed that miR-224-5p expression increased in the ARC group. Moreover, transfection of miR-224-5p mimics or inhibitors decreased or increased PLCD3-OT1 expression levels. To sum up, these data are conformed with our hypothesis and suggest that PLCD3-OT1 may interact with miRNA-224-5p to link miRNA-224-5p and the posttranscriptional network in ARC pathogenesis. 

The results of the bioinformatics analysis showed that PLCD3-OT1 overlapped PLCD3 on the sense strand. PLCD3, a member of the PLCs (phospholipases), is a key enzyme in phosphoinositide turnover. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP₂) to generate two second messengers, inositol 1,4,5-trisphosphate (IP₃), and diacylglycerol (DAG),⁴² which play important roles in the cell cycle, proliferation, apoptosis, and cell movement. IP₃ promotes the activation of protein kinase C (PKC), and DAG can regulate the release of Ca²⁺ from intracellular stores.^43,44 In previous studies, PLCD3 was shown to play important roles in numerous biological processes, such as the development of trophoblasts and cardiomyocytes, promoting neurite expansion, and maintaining normal heart function.^45–47 Researches concerning PLCD3 in epithelial cells are rare. To the best of our knowledge, only a few studies have shown that PLCD3 can promote the proliferation and migration of neoplastic mammary epithelial cells and nasopharyngeal carcinoma cells.^48,49 Furthermore, whether PLCD3 is involved in oxidative stress in cells has been investigated in a few studies, which demonstrated a protective effect of PLCD3 on oxidative damage of cardiomyocytes.^47,50 Oxidative stress can be regarded as an imbalance between ROS and antioxidant defense, oxidant production, and the state of glutathione redox buffers in general. However, a new paradigm of redox signaling has emerged, and in such situation the function of ROS and oxidants can be considered as intracellular signaling molecules, where ROS- and oxidant-induced death signaling is converted into survival signals.^51–54 Therefore, to confirm that PLCD3 plays a part in the oxidative damage of LECs, we measured the expression levels of PLCD3 in the ARC lens capsule and found that they were increased. Furthermore, we tested the expression levels of PLCD3 and PLCD3-OT1 by establishing a model of oxidative damage of LECs. The results showed that PLCD3 and PLCD3-OT1 were highly expressed in this model. Then, we silenced PLCD3 by RNA interference, and the results were entirely consistent with our hypothesis that PLCD3 knockdown inhibited LECs proliferation and increased cell apoptosis after UVB exposure. We also tested the expression levels of PLCD3 after overexpression of PLCD3-OT1, PLCD3-OT1 expression levels when silencing PLCD3, and the expression levels of PLCD3-OT1 and PLCD3-OT1 after transfection with miRNA 224-5p mimics or inhibitors. We found that the expression levels of PLCD3-OT1 and PLCD3 were closely related. 

In summary, we demonstrated that PLCD3 plays a protective role in the oxidative damage of LECs and that PLCD3-OT1, as a ceRNA, regulates PLCD3 expression by sponging miR-224-5p in ARC. However, the mechanism through which PLCD3 regulates ARC progression and the reason for decreased PLCD3-OT1 in ARC patients remain unclear. Although we analyzed the potential protein–protein interaction by bioinformatics analysis of the mRNA profile (Supplementary Fig. S2), further studies will use human lenses at different stages of cataract development and use the models with different degrees of oxidative damage to clarify the cause and effect relationship between PLCD3-OT1 changes and cataract formation, and the potential protein–protein interaction was also verified. 

Acknowledgments

Supported by grants from the National Natural Science Foundation of China (No. 81770906, No.81873676, No.81974129; Beijing, China) and the Science and Technology Project of Nantong Municipality, Jiangsu Province, China (QA2019061; Nantong, Jiangsu, China). 

Disclosure: J. Xiang, None; Q. Chen, None; L. Kang, None; G. Zhang, None; Y. Wang, None; B. Qin, None; J. Wu, None; T. Zhou, None; Y. Han, None; H. Guan, None